(1) Concomitant with activation by Ca2+ and calmodulin, the calmodulin-dependent phosphoprotein phosphatase was found to undergo a time-dependent deactivation process. The deactivation is a first-order reaction and is accelerated ty the presence of substrate and competitive inhibitors. It can be reversed or prevented by addition of divalent metal ions like Ni2+, Mn2+, etc., which have been shown previously to activate the enzymatic activity. This study indicates that the phosphatase is not simply a Ca2+ calmodulin-stimulated enzyme. It requires at least one additional metal ion for structural stability. (2) Examination of the mechanism of deactivation of the calmodulin-dependent protein phosphatase led to the discovery that the enzyme contains 0.2-0.6 mol of phosphate per mol of enzyme. It also contains nearly stoichiometric amounts of Fe3+ and Zn2+. (3) The minimum number of Ca2+ ions needed for the activation of calmodulin-dependent protein phosphatase was found to be 3.5 and 1.7 in the absence and presence of Mg2+. The observation suggests that the presence of the activating metal ion enhances the affinity of the enzyme for calmodulin not saturated with Ca2+. (4) The activation of calmodulin-dependent protein phosphatase by Ni2+ ions is a time-dependent, first-order process. Like the deactivation reaction, the presence of substrate accelerates the activation. Interactions among the active-site, the metal ion binding, and the calmodulin binding domain are implicated. (5) A Ca2+ inhibited protein phosphatase has been partially purified from bovine brain. The enzyme catalyzes the dephosphorylation of two phosphorylated sites on synapsin I selectively. The results indicate that this phosphatase and the calmodulin-dependent phosphatase may function in vivo in a coordinated manner.